The Role and Mechanism of Perilla frutescens in Cancer Treatment

Perilla frutescens is an annual herb of the Labiatae family and is widely grown in several countries in Asia. Perilla frutescens is a plant that is used medicinally in its entirety, as seen in its subdivision into perilla seeds, perilla stalks, and perilla leaves, which vary more markedly in their chemical composition. Several studies have shown that Perilla frutescens has a variety of pharmacological effects, including anti-inflammatory, antibacterial, detoxifying, antioxidant, and hepatoprotective. In the absence of a review of Perilla frutescens for the treatment of cancer. This review provides an overview of the chemical composition and molecular mechanisms of Perilla frutescens for cancer treatment. It was found that the main active components of Perilla frutescens producing cancer therapeutic effects were perilla aldehyde (PAH), rosmarinic acid (Ros A), lignan, and isoestrogen (IK). In addition to these, extracts of the leaves and fruits of Perilla frutescens are also included. Among these, perilla seed oil (PSO) has a preventive effect against colorectal cancer due to the presence of omega-3 polyunsaturated fatty acids. This review also provides new ideas and thoughts for scientific innovation and clinical applications related to Perilla frutescens.


Introduction
Cancer is one of the world's leading diseases in terms of mortality [1], the second leading cause of death, and a major public health problem worldwide [2,3]. As of 2019, prostate cancer, colorectal cancer, liver cancer, and lung cancer are the four types with the highest incidence and mortality rates in men, while breast cancer and cervical cancer are the most common among female cancer patients, according to related research studies [4]. The incidence and mortality of cancer in different regions are significantly different [1,3], which is closely related to the factors leading to cancer occurrence. There have been many literature reports on the risk factors for cancer, including diet, lifestyle, family genetic history, ionizing radiation, and other nine important categories [2,5]. In addition to the more common cancer treatment methods such as surgery, chemotherapy, and radiotherapy [6], the current effective cancer treatment methods also include gene therapy [7,8] and immunotherapy [9].
Based on the existence of drug resistance and toxicity, scientists are actively looking for herbs or plant metabolites that can have anticancer potential [10,11]. Numerous studies have shown that chemicals derived from plants can have preventive and therapeutic effects on cancer [12]. Flavonoids [13,14], natural phenolic compounds from herbs and dietary plants [15], polyphenols and their compositions [16,17], and dietary unsaturated fatty acids [18] all have preventive and anti-tumor effects. Bioactive compounds derived from plants, such as tanshinone, astragaloside, berberine, ginsenoside, and matrine, can inhibit tumor growth, metastasis, and

Network Diagram of Anti-Tumor Effect of Perilla frutescens
In order to confirm the anti-tumor effect of Perilla frutescens, a network pharmacological analysis of Perilla frutescens was carried out based on its multi-component, multi-target, and multi-level properties. According to OD ≥ 0.3 and DL ≥ 0.18, 13 active components (see Supplementary File S1 for details) and 144 related proteins were screened from the TCMSP database (https://old.tcmsp-e.com/tcmsp.php (accessed on 11 May 2023)). The obtained protein name was entered in the Multiple Proteins section of the STRING database (https: //cn.string-db.org (accessed on 12 May 2023)), and the Homo sapiens species was selected. Download the obtained gene name and match it with the protein name. Topological network maps of the 13 active components and 144 corresponding genes were constructed using Cytoscape software 3.9.1 (see Supplementary File S2 for details). In the figure, the plant name is the green module, the orange template is the active ingredient, and the red module is the corresponding gene. KEGG and GO analysis was performed on 144 target genes through the DAVID database (https://david.ncifcrf.gov (accessed on 12 May 2023)). The KEGG results showed that nine of the top 30 signaling pathways were associated with cancer. GO analysis showed that it was closely related to apoptosis, migration, proliferation, and other related processes (see Supplementary File S3 for details). It mainly includes a series of cancer-related signaling pathways, such as lung endometrial cancer, breast cancer, non-small cell lung cancer, pancreatic cancer, gallbladder cancer, and bladder cancer, as shown in Figure 2. These signaling pathways demonstrate that the relevant active components of Perilla frutescens can produce anti-tumor effects through these cancer-related pathways.

Network Diagram of Anti-Tumor Effect of Perilla frutescens
In order to confirm the anti-tumor effect of Perilla frutescens, a network pharmacological analysis of Perilla frutescens was carried out based on its multi-component, multi-target, and multi-level properties. According to OD ≥ 0.3 and DL ≥ 0.18, 13 active components (see Supplementary File S1 for details) and 144 related proteins were screened from the TCMSP database (https://old.tcmsp-e.com/tcmsp.php (11 May 2023)). The obtained protein name was entered in the Multiple Proteins section of the STRING database (https://cn.string-db.org (12 May 2023)), and the Homo sapiens species was selected. Download the obtained gene name and match it with the protein name. Topological network maps of the 13 active components and 144 corresponding genes were constructed using Cytoscape software 3.9.1 (see Supplementary File S2 for details). In the figure, the plant name is the green module, the orange template is the active ingredient, and the red module is the corresponding gene. KEGG and GO analysis was performed on 144 target genes through the DAVID database (https://david.ncifcrf.gov (12 May 2023)). The KEGG results showed that nine of the top 30 signaling pathways were associated with cancer. GO analysis showed that it was closely related to apoptosis, migration, proliferation, and other related processes (see Supplementary File S3 for details). It mainly includes a series of cancer-related signaling pathways, such as lung endometrial cancer, breast cancer, nonsmall cell lung cancer, pancreatic cancer, gallbladder cancer, and bladder cancer, as shown in Figure 2. These signaling pathways demonstrate that the relevant active components of Perilla frutescens can produce anti-tumor effects through these cancer-related pathways.

Active Ingredients
As an important annual herb in the Labiaceae family, Perilla frutescens has rich chemical constituents and biological functions and is widely used in food and medicine fields [44]. Related studies have found that the active components of Perilla frutescens can be divided into 14 main active components (see Figure 3). The detailed composition is shown in Table 1.

Active Ingredients
As an important annual herb in the Labiaceae family, Perilla frutescens has rich chemical constituents and biological functions and is widely used in food and medicine fields [44]. Related studies have found that the active components of Perilla frutescens can be divided into 14 main active components (see Figure 3). The detailed composition is shown in Table 1.

Alkaloids
Alkaloids exist in a variety of traditional Chinese herbs; for example, hyoscypane alkaloids [45] are mainly found in Solanaceae, dibenzyl isoquinoline alkaloids [46] are mainly found in the seeds and fruits of lotus, and Evodia alkaloids are mainly found in Evodia officinalis of the rutaceae [47]. In recent years, the presence of the alkaloid compound neoechinulin A in Perilla frutescens has been found, which can produce anti-inflammatory effects on RAW267.4 cells stimulated by lipopolysaccharide.

Phenylpropane
Phenylpropane compounds are secondary metabolites of plants derived from phenylalanine, an aromatic amino acid in most plants, or tyrosine in some monocotyledon plants. Some studies have found that Perilla frutescens collected from Taiwan contained elemin, a phenylpropanoid component, while this component was not detected in Japanese Perilla frutescens [48]. Some studies have found that phenylpropanoid compounds can be separated from the ethanol extract of Perilla leaves, including allyl tetramethoxybenzene, elietin, and myristin, among which elietin and myristin have been found to inhibit the production of pro-inflammatory cytokines in pneumonia in a concentrationdependent manner within a certain concentration range [49].

Terpenoids
Terpenoids are the most common compounds in Perilla frutescens, of which the monoterpenoid PAH is the main component of Perilla leaf essential oil, which can improve the in vivo function of intestinal inflammation through JNK-mediated cytokine ajay [50]. At the same time, cytoplasmic DNA-induced innate immune responses can be inhibited by inhibiting cGAS activity [51]. The triterpenoid camelliol C [52] was identified from Perilla

Alkaloids
Alkaloids exist in a variety of traditional Chinese herbs; for example, hyoscypane alkaloids [45] are mainly found in Solanaceae, dibenzyl isoquinoline alkaloids [46] are mainly found in the seeds and fruits of lotus, and Evodia alkaloids are mainly found in Evodia officinalis of the rutaceae [47]. In recent years, the presence of the alkaloid compound neoechinulin A in Perilla frutescens has been found, which can produce antiinflammatory effects on RAW267.4 cells stimulated by lipopolysaccharide.

Phenylpropane
Phenylpropane compounds are secondary metabolites of plants derived from phenylalanine, an aromatic amino acid in most plants, or tyrosine in some monocotyledon plants. Some studies have found that Perilla frutescens collected from Taiwan contained elemin, a phenylpropanoid component, while this component was not detected in Japanese Perilla frutescens [48]. Some studies have found that phenylpropanoid compounds can be separated from the ethanol extract of Perilla leaves, including allyl tetramethoxybenzene, elietin, and myristin, among which elietin and myristin have been found to inhibit the production of pro-inflammatory cytokines in pneumonia in a concentration-dependent manner within a certain concentration range [49].

Terpenoids
Terpenoids are the most common compounds in Perilla frutescens, of which the monoterpenoid PAH is the main component of Perilla leaf essential oil, which can improve the in vivo function of intestinal inflammation through JNK-mediated cytokine ajay [50]. At the same time, cytoplasmic DNA-induced innate immune responses can be inhibited by inhibiting cGAS activity [51]. The triterpenoid camelliol C [52] was identified from Perilla seed species, and a series of pentacyclic triterpenes were discovered, including ursolic acid [53], oleanolic acid, corosolic acid, 3surface acid, marlinic acid, and 3-surface equine linolenic acid [54], all of which have anti-tumor effects [55].

Flavonoids
Perilla leaves are composed of many types of active ingredients, but mainly flavonoids. Studies have confirmed that it acts as an anti-inflammatory agent in vivo and in vitro in specific dermatitis models [57]. The most common compound of Perilla frutescens flavonoids is luteolin, which has been confirmed to have anti-inflammatory, anti-itch [58], anti-allergic [59], anti-cytotoxic [60], and antibacterial [43] activities.

Anthocyanins, Coumarins, Carotenoids, and Neolignans
Anthocyanin pigments are the main cause of red Perilla leaves [61]. Two new lignans identified in Perilla frutescens, magnosalin and andamanicin, can act as inhibitors of tumor necrosis factor and nitric oxide synthesis in RAW264.7 cells induced by lipopolysaccharide [32]. Aesculin, as a coumarin, was first discovered in Perilla frutescens and has been found to have a certain relationship with anti-inflammatory effects [30].

Glucoside and Peptide
Twelve secondary metabolites isolated from perillafrutoside A, perillafrutoside B, and ten other known compounds were found in perillafrutoside A, among which perillafrutoside A can inhibit the growth of Enterococcus faecalis [72]. Monoterpene glucosides, perillosides A and C, obtained from perilli leaves, have also been found to be aldose reductases. One of the richest sources of peptides from Perilla seeds, peptides obtained from Perilla seeds can improve muscle synthesis and motor performance in mice [73]. Two novel antioxidant peptides were purified and identified from Perilla seeds to inhibit lipid peroxidation in the liver [74].

Anti-Cancer Compound Structural Formula
Related studies have found that Perilla frutescens mainly generates anti-tumor activity against liver cancer, lung cancer, and breast cancer through a series of related mechanisms such as PAH [88], IK [89], luteolin [90], Ros A [91], ethanol extract of Perilla leaves [92], Perilla extract [93], PO [94], etc. The structural formula of related compounds is shown in Figure 4.

Anti-Cancer Effect
Inducing cell apoptosis, blocking the cell cycle, reducing cell inflammation and oxidative stress, inhibiting cell metastasis, growth, and proliferation, and inducing cell senescence are the main pathways and phenotypes of the anti-tumor effects of Perilla frutescens, as shown in Table 2.

Anti-Cancer Effect
Inducing cell apoptosis, blocking the cell cycle, reducing cell inflammation and oxidative stress, inhibiting cell metastasis, growth, and proliferation, and inducing cell senescence are the main pathways and phenotypes of the anti-tumor effects of Perilla frutescens, as shown in Table 2.

Cell Transfer
Metastases are a hallmark of cancer and cause the largest number of cancer-related deaths [95]. Epithelial-mesenchymal transformation (EMT) is a process by which epithelial cells acquire mesenchymal characteristics. In cancer, EMT is associated with tumor occurrence, invasion, metastasis, and treatment resistance [96]. Relevant studies have shown that isoproterenol (ISO) increases the migration and invasion of MDA-MB-231 human breast cancer cells and Hep3B human hepatocyte cancer cells [97]. The ethanol extract of Perilla leaf can reverse cancer cell metastasis induced by adrenergic agonists through the SRCT231F-mediated EMT pathway [92]. PAH is one of the active components of Perilla frutescens, which can affect prostate cancer-induced bone metastasis by inhibiting the NF-κB pathway [98].

Apoptosis
Apoptosis is an orderly and coordinated cellular process that occurs under physiological and pathological conditions. The mechanism of apoptosis is complex and involves many pathways. Defects can occur at any point in these pathways, leading to malignant transformation of affected cells, tumor metastasis, and anti-cancer drug resistance [99]. Ethanol extract of Perilla leaf can inhibit the growth of HCT116 and H1299 cells in a dosedependent manner, inhibit cell colony formation, increase the G1 cell population, change nuclear morphology, and induce cell apoptosis [100]. The YAP/WW domain contains transcription factors (TAZ) that are critical for cell proliferation, survival, and self-renewal. It has also been shown to have an important carcinogenic effect on various tumors [101]. Perilla leaf extract (PLE) can induce phosphorylation of YAP/TAZ, resulting in its inactivation, and thus produce anti-tumor effects. The results suggest that PLE inhibits cell growth and increases apoptosis in breast cancer (BC) cells by inactivating YAP activity in a LATS1/2-dependent manner [102]. In the treatment of melanoma cells, IK can produce ROS, up-regulate the expression of Bax and Bcl-2, inhibit the growth of melanoma cells, and induce apoptosis.

Cell Senescence
In most species, aging may induce a number of degenerative diseases characterized by a debilitating loss of tissue or cell function [105]. However, for the aging of cancer cells, its basic feature is stable proliferation arrest induced by various stressors [106]. PDMF can induce senescence in A549 human adenocarcinoma cells through the p21-p549 pathway but has no effect on normal bronchial epithelial cells [107].

Oxidative Stress Response and Cellular Inflammation
Tumor necrosis factor TNF-α is a major inflammatory cytokine that is particularly important in the development of tumors [108]. Endothelial microparticles are important factors in inflammation-related diseases. Studies have found that phenolic compounds contained in ethyl acetate and ethanol extracts extracted from Perilla fruit can reduce endothelial microparticles induced by TNF-α, thereby protecting endothelial cells from vascular inflammation [109]. Perilla extract can improve colitis induced by sodium dextran sulfate (DSS) in mice by inhibiting the expression of inflammation-related proteins such as COX-1. NF-κB and STAT3 are major transcriptional regulators of inflammatory signaling. Perilla extract inhibits DDS-induced NF-κB and STAT3, thereby reducing pro-inflammatory signaling [93]. Oxidative stress is a state caused by disruption of the balance between ROS production and antioxidant defense [110]. The Ros A component in PSO can reduce the production of ROS in the A549 cell line and the mRNA levels of related IL-6, IL-8, COX-2, etc., resulting in decreased expression of TNF-α induced NF-κB, JNK, MnSOD, and FOXO1 signaling pathways [91]. The ethyl acetate and ethanol extracts of Perilla frutescens can inhibit the production of ROS and have a protective effect on lipid peroxidation, indicating their potential to protect against oxidative stress in liver diseases [111].

Cell Growth
Cell growth is one of the key markers in cancer. Amp-activated protein kinase (AMPK) is associated with autophagy in unused tissues. PAH can activate AMPK by increasing phosphorylation at THr172, resulting in the increase of AMPK-related proteins such as caspase-3 and p53, resulting in increased autophagy levels and inhibiting the growth of gastric cancer [112].

Cell Proliferation
The abnormal proliferation of cancer cells is an important sign of a tumor and also an important reason for the expansion of cancer cells' colonies. The PI3K/AKT signaling pathway is one of the frequently activated signaling pathways in the process of cancer, which is closely related to the occurrence and development of tumors. Studies have shown that IK isolated from Perilla extract can inhibit liver cancer (HCC) tumor proliferation by inhibiting pAKT levels without affecting total AKT levels and blocking the PI3K/AKT pathway [113].  PO enriched with n-6 and n-3 PUFA altered the membrane fatty acid composition of the liver and inhibited the development of hepatocellular carcinoma in rats. [118] Luteolin Colon cancer HT-29 human colon cancer cells Dose: 0-60 µmol/L By activating caspase-3, -7, and -9, the cleavage of poly (ADP-ribose) polymerase was enhanced, the expressions of p21 (CIP1/WAF1), survivin, Mcl-1, Bcl-x(L), and Mdm-2 were decreased, and the activities of cyclin-dependent kinase (CDK) 4 and CDK2 were inhibited.

Summary of Anticancer Mechanism
Perilla frutescens and its active components or derivatives mainly produce anti-tumor effects on cell growth, proliferation, inflammation, cycle, apoptosis, and metastasis through ROS, NF-κB, PI3K/AKT, JNK, and other pathways, as shown in Figure 5.

Summary of Anticancer Mechanism
Perilla frutescens and its active components or derivatives mainly produce anti-tumor effects on cell growth, proliferation, inflammation, cycle, apoptosis, and metastasis through ROS, NF-κB, PI3K/AKT, JNK, and other pathways, as shown in Figure 5.

Preventative Effects
Relevant studies have shown that unsaturated fatty acids can be used as adjuvant therapeutic agents in cancer treatment [123]. Omega-3 polyunsaturated fatty acids (PUFAs) are considered immune nutrients and are commonly used in the nutritional treatment of cancer patients due to their rich biological effects [124]. The intake of such dietary oils is particularly important for human health. PO is a complex of unsaturated fatty acids from Perilla. PO is rich in the omega-3 polyunsaturated fatty acid alpha-linolenic acid, which can effectively reduce the risk of colon cancer [125,126] Table 3.

Summary and Outlook
Cancer is a kind of malignant disease that is difficult to treat, not only because of its diversification in proliferation and metastasis but also because cancer cells have strong adaptability [130]. Chinese herbs are an effective source of adjuvant cancer treatment and have been found to treat or prevent cancer in a variety of ways. Relevant studies have shown that various plant extracts and plant active ingredients can activate various pathways in cancer cells, including apoptosis [131]. Phenolic compounds extracted from herbs can inhibit or weaken the occurrence, progression, and metastasis of cancer [132]. Artemisinin and its derivatives have the therapeutic potential to induce iron death in cancer cells [133]. Many studies have shown that plants such as garlic, olives, and pomegranates are effective in preventing colon cancer [134].
Prior to writing this review, relevant information was consulted. Related reviews were searched in PubMed with the keywords "Perilla frutescens," "Perilla frutescens and caner," "Perilla frutescens and carcinoma," "Perilla frutescens frutescens," and "Perilla frutescens and tumor." Few reviews were found in the last 5 years, and none were related to cancer treatment. Among the relevant reviews that have been reviewed, there are three related to the pharmacological and phytochemical effects of Perilla frutescens, which mainly summarize the phytomedicinal, ethnobotanical, phytochemical, and pharmacological effects of Perilla frutescens [24,135,136]. Three reviews were related to the active components of Perilla frutescens, namely, perillone and IK [137], Ros A [138], and PAH [88]. The review of Ros A gives an overview of its anti-cancer potential, but Ros A is derived from a variety of herbs, including rosemary and Perilla frutescens.
In this review, the active components of Perilla frutescens were summarized according to the relevant literature and the reading summary of the literature. The anticancer effects of related targets were demonstrated by biogenic analysis. The molecular mechanism and preventive effect of the components of Perilla frutescens with anticancer activity, including PAH, Ros A, luteolin, PO, etc. This review also gave a general description of Perilla frutescens's treatment of cancer-related phenotypes, such as cell proliferation, cell metastasis, cell cycle, etc. It was found that Perilla frutescens mainly targeted the cellular inflammation and oxidative stress responses of cancer cells as the main targets to produce anticancer activity. In the process of writing the review, it was found that there were no relevant studies on the attenuated and synergistic effects of Perilla frutescens as an adjuvant therapy for cancer. Perilla frutescens contains many kinds of effective components, but there are few anticancer studies on PAH, PSO, perillone, and so on, which are unique to Perilla frutescens, and most studies focus on Ros A and IK.
Through the literature review, it was found that Perilla frutescens has a unique active ingredient, PO, which can prevent cancer. Through the systematic summary of PO, it was found that it is rich in omega-3 polyunsaturated fatty acids, which can have a positive effect on human health and prevent the occurrence of some diseases. Starting from this basis, according to these properties of PO, health care products and drugs can be developed to improve the quality of life, prevent major diseases, and make significant contributions to human health.  Data Availability Statement: All data generated or analyzed during this study are included in this paper, and further inquiries can be directed to the corresponding author (E-mail: 20080017@nxmu.edu.cn).